Histidinohydroxylysinonorleucine (HHL) is a trivalent type of cross-link in skin, which provides collagen for stability and other physiological properties. We established a novel highly sensitive method to measure HHL by using reversed phase HPLC after labeling skin hydrolysates with 9-fluorenylmethyl chrolformate. We measured the content of HHL in the skin specimens of patients with systemic sclerosis and compared it with the HHL of age- and site-matched control skin specimens using this method. Half of the skin sample was processed for a conventional histological examination and chose skin samples that showed the characteristic histological change of SSc, thickened and hyalinized collagens in the entire dermis. The content of HHL in the SSc was significantly increased compared with that of controls when expressed as the molecular ratio of HHL to collagen. The increased amount of HHL in collagen molecules may be associated with the pathological fibrosis of the disease. It is worth inv
… Moreestigating if this increase is specific for SSc or not, or which facto (s) regulates the formation of intermolecular cross-links in vivo.We investigated the effects of hypoxia and low serum concentartion on the expression of extracellular matrix-related genes and the formation of trifunctional intermolecular cross-link of type I collagen by dermal fibroblasts. In the studies of reverse transcriptase polymerase chain reaction (RT-PCR), both hypoxia and low serum concentration revealed the increased mRNA expression of transforming growth factor-beta and the decreased mRNA expression of metalloproteinase-1 in the monolayr culture of dermal fibroblasts from normal individuals and patients with systemic sclerosis. In the three-dimensional culture supplemented with ascorbic acid 2-phosphate, hypoxia remarkably suppressed the formation of histidinohydroxylysinonorleucine, a mature type of trifunctional intermolecular cross-link of type I collagen.The altered mRNA expression of transforming growth factor-beta and metalloproteinase- 1 may well account for the pathoetiology of the post inflammatory stage of systemic sclerosis. The suppressed formation of histidinohydroxylysinonorleucine under the culture condition of hypoxia or low serum concentartion disagreed with the previous in vivo finding. Further studies are required to elucidate factor (s) to regulate the formation of trifunctional intermolecular cross-link of type I collagen. Less